The present invention relates to human bile salt-stimulated lipase (BSSL) obtainable from transgenic sheep. The invention further relates to transgenic sheep whose germ cells and somatic cells contain a recombinant nucleotide molecule comprising a nucleotide sequence encoding for human BSSL. The invention also relates to methods for producing said transgenic animals, as well as to methods for producing human BSSL derived from transgenic animals. In addition, the invention provides the use of compositions comprising BSSL in the treatment of diseases relating to exocrine pancreatic insufficiency, and for improvement of the utilization of dietary lipids in preterm born infants.
Human Bile Salt-Stimulated Lipase
Bile Salt-Stimulated Lipase (BSSL) is the major lipolytic activity present in human milk (Wang and Johnson, 1983; Wang and Hartsuck, 1993). As its name implies, the enzyme is not active in the milk but is activated in the intestine by bile salts. In mammals a similar enzyme activity is also secreted from the pancreas into the intestine. The cDNA sequences for both the mammary and pancreatic enzyme are identical indicating that they are the product of a single gene (Reue et al., 1991; Lidberg et al., 1992). BSSL shares with other pancreatic lipases a triacylglycerol hydrolase activity but BSSL is the only intestinal lipase which hydrolyses cholesterol ester and other fatty acid esters, such as vitamin A ester. The protein is stable to both proteases and the acid environment in infant stomachs. These unique activities and the large quantity of BSSL in human milk suggest that this enzyme is physiologically important.
The cDNA sequence (SEQ ID NO: 2) and deduced amino acid sequence (SEQ ID NOS: 3and 4) of BSSL have been disclosed by Nilsson et al. (1990); in U.S. Pat. No. 5,200,183 (Oklahoma Medical Research Foundation); and in WO 91/18923 (Astra AB). Human BSSL is a glycoprotein consisting of 772 amino acids. The C-terminal portion is composed of 16 repeats each of 11 amino acids having consensus PVPPTGDSGAP (SEQ ID NO: 5). The genomic DNA sequence (SEQ ID NO: 1) encoding human BSSL is disclosed by Lidberg et al. (1992) in U.S. Pat. No. 5,616,483 (Astra AB).
BSSL is heavily glycosylated, with one potential N-linked glycosylation site (Asp187) and two potential O-linked sites per repeat (Baba et al., 1991). Even though the glycosylation state of the enzyme is not critical for activity in vitro (Hansson et aL, 1993), O-glycosylation nevertheless appears important for the biological function of BSSL. Loomes et al. (1997) have suggested that the C-terminal repeat region is important for prevention of non-specific hydrophobic interactions and that it counteracts a tendency of BSSL to self-aggregate. Wang et al. (1995) have shown that the repeat region contains different Lewis antigens, suggesting that it may also contribute to adhesive activity in the physiological function of BSSL. Spilburg et al. (1995) and Bosner et al. (1988) have suggested that the C-terminal domain may be important for heparin binding and interactions with proteoglycans on cell surfaces. Immunohistochemical experiments on mouse, rat and human tissues have indicated binding of the BSSL protein to intestinal cell membranes. O-glycosylation might thus be important for such binding of BSSL to a xe2x80x9cBSSL-receptorxe2x80x9d in the intestine and for proper physiological function of the enzyme.
The expression of both cDNA and genomic constructs for BSSL, from the murine whey acidic protein (WAP) promoter, in the milk of transgenic mice have been disclosed (Strxc3x6mqvist et al., 1995). Levels of up to 1 mg/ml were obtained in milk and the protein was 100% active in vitro. However, the transgenic form of the enzyme exhibited a considerably reduced glycosylation state when compared to native enzyme or enzyme produced in cell culture. Recombinant human BSSL derived from transgenic mice apparently lacked the O-glycosylation pattern of native human enzyme.
Expression of Heterologous Proteins in the Milk of Transgenic Animals
The cloning and characterization of the ovine xcex2-lactoglobulin (BLG) gene has been disclosed by Ali and Clark, 1988. It was demonstrated that BLG is consistently expressed at high levels in the milk of mice made transgenic for the entire gene (Simons et al., 1987; Harris et al., 1991). Further experiments demonstrated that the BLG promoter region can direct high levels of expression of a heterologous human protein to the milk of transgenic mice (Archibald et al., 1990). The generation of transgenic sheep, expressing human proteins in their milk using the BLG gene to drive expression, indicated that this technology might be viable as a commercial production route (Simons et al., 1988; Clark et al., 1989). The feasibility of applying this technology to the modification of livestock milks has been confirmed, demonstrating high level expression of human alpha-1-antitrypsin in the milk of transgenic sheep (Wright et al., 1991; Carver et al., 1992; Carver et al., 1993; Cooper and Dalrymple, 1994). This high level expression of a heterologous protein in livestock milk was the result of using a fusion of the BLG promoter region to human genomic sequences (Wright et al., 1991). The vector pMAD6, disclosed in U.S. Pat No. 5,639,940, has been designed to express genes from the BLG promoter.